Oral Fluid Collection Enhances Testing

Iowa State University (ISU) swine disease ecologist Jeff Zimmerman, DVM, merely chuckles at the friendly criticism he received five years ago when he submitted his first proposal to evaluate pig “oral fluid” as a potential surveillance method for early detection of porcine reproductive and respiratory syndrome (PRRS) virus

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Iowa State University (ISU) swine disease ecologist Jeff Zimmerman, DVM, merely chuckles at the friendly criticism he received five years ago when he submitted his first proposal to evaluate pig “oral fluid” as a potential surveillance method for early detection of porcine reproductive and respiratory syndrome (PRRS) virus.

“Oral fluids are different than saliva,” Zimmerman explains. “Saliva comes from the salivary glands, and oral fluids contain saliva, but they also contain fluid that comes from the capillaries or the blood system.”

The skepticism has died down, and since 2005, Zimmerman has received substantial grants to pursue his work. In the process, pig oral fluid has continued to gain credence as a viable alternative to blood testing for screening pigs for PRRS virus.

History of Oral Fluid

Oral fluid use has gained special traction in recent years as a quick and safe way to test patients for HIV.

“Testing for pathogens using oral fluid does seem kind of far-fetched until you look in the literature and see how much work has been going on in this area since 1909,” Zimmerman remarks.

For Zimmerman and his colleagues at the ISU College of Veterinary Medicine in Ames, the search for a new test collection method began in March 2005, when his team had grown weary of collecting tonsil scrapings in a long-term study of PRRS persistence.

“Pretty much out of desperation, I took some ropes and hung them in pens. The pigs very happily chewed and played with the ropes. We found it was easy to recover oral fluid samples, and they provided some surprisingly promising diagnostic data,” he says. That data was first reported in December 2005 at the International PRRS Symposium in St. Louis, MO.

Tools Needed

To collect oral fluids, Zimmerman uses three-strand, 100% cotton rope, unbleached and untreated. The rope is readily available over the Internet in spools, at a cost of about 40¢/ft. He prefers ½-in.-thick rope for weaned pigs and ⅝-in. rope for larger pigs.

To collect the sample, he suggests using just enough rope so that it drapes down to about shoulder height on a pig. The rope is hung in a pen for about 20 minutes. Typically, 75% of the pigs in a pen will chomp on the rope in that span of time, providing suitable samples.

Next, place the rope in a plastic bag, squeeze out the fluids, pour into a tube and send to a diagnostic laboratory for testing. Standard serum “snap cap” tubes can be used, and samples should be kept chilled or frozen — just like serum samples.

The oral fluid collection process is much simpler than the tedious task of singling out and snaring pigs to collect blood for testing. In a PRRS study that can include numerous blood tests, pigs quickly become leery of the whole process, Zimmerman attests. Oral fluid testing also offers a much safer and more biosecure means of collecting samples from breeding stock.

In people, oral fluid testing for HIV has set the standard, yielding near 100% accuracy. “Test development and evaluation is our current focus. So far, the data shows that oral fluids are at least as accurate as blood samples, and may be even better for detecting PRRS virus in populations of pigs. We think this particular technique will be most useful in surveillance,” he stresses. “One of the biggest challenges we have as producers and practitioners is monitoring the circulation of infectious diseases in our herds.”

The oral fluid protocol facilitates rapid, easy testing and turnaround time for monitoring PRRS virus. Other researchers are also studying its application for monitoring influenza, mycoplasma and porcine circovirus infections in pig populations.

Preliminary results indicate that oral fluids provide similar results to blood testing for PRRS using PCR, Zimmerman says. In his studies, there was 100% agreement at Day 7 and, farther out, more PRRS positives were found in oral fluids than in serum collection.

“Over time, we have found that there is better detection of PRRS virus by PCR in oral fluids than there is in serum,” he points out. In studies just completed, 90% of collections were positive for PRRS virus by oral fluids 21 days after infection, compared to 80% with serum.

“One question that always comes up relates to the detection of early infections. What is the chance of finding that one PRRS-positive pig in a pen?” he asks.

To test that question using oral fluid samples in a PRRS-negative commercial finisher, one pig was removed from each of 36 pens, moved to another site, penned together and vaccinated for PRRS. Four days later, one pig was moved back into each of those 36 occupied pens. Each pen held 26 head. Out of those 36 pens, 24 pens (67%) of the groups turned up PRRS-positive when one oral fluid sample from each pen was tested by PCR.

“The results were absolutely phenomenal. You would never get that level of detection if you tested one blood sample from each pen,” Zimmerman emphasizes.

Recent work by ISU researchers John Prickett and Apisit Kittawornrat on detection of PRRS antibodies in oral fluids has shown oral fluid detection of the PRRS virus at Day 9 on an assay, compared with an average of 14-17 days on detection by serum collection. Their work could provide a cheaper alternative to PCR testing for surveillance of PRRS virus, Zimmerman suggests.

Oral Fluid vs. Blood

Another common question is, are more pathogens detectable in oral fluid or blood?

“I think we are going to find pathogens in oral fluids that you won’t find in blood — which makes oral fluid a very valuable diagnostic tool. This has a huge potential to provide new tools for swine health, but there is a lot of work yet to be done,” Zimmerman says.

Other Applications

“Oral fluid collection is a new tool that is easily adaptable to a lot of situations,” says Rodney “Butch” Baker, DVM, senior clinician in Veterinary Diagnostic and Production Animal Medicine at Iowa State University.

Plans are to use oral fluid as a big part of the epidemiology aspects of the startup PRRS pilot project in Iowa County, IA, to evaluate area spread of the virus.

“We feel that once the industry decides to eliminate PRRS, that oral fluid is going to be a valuable tool for determining the status of pigs before they move. We have 23 million pigs coming into Iowa annually,” he notes.

Because multiple testing is easy with oral fluid, producers can repeat testing during late nursery into grow-finish to pinpoint when PRRS virus is surfacing and determine the status of the disease in a population of animals. Then, action can be taken to control movement as needed, a vital step in a regional control or eradication program, Baker says.

In the future, oral fluid collection could provide support to individual serum sampling to determine that hog exports are free of a number of infectious diseases, he adds.

Producer, Pig Training

With minimal training, producers can learn how to set up oral fluid collection in their barns, and collect and submit oral fluid samples to the lab with the assistance of their swine veterinarian, Baker explains.

Boars require some training before oral fluid collection. Place the rope on the floor so they can play with it for 20 minutes or so until they realize it is non-threatening.

It doesn’t take more than a day or two to train young weaned pigs in a commercial setting. Pigs 7-8 weeks old through finishing age easily figure out the process.

Don’t leave the ropes in pens longer than 20 minutes, he advises. Pigs will become bored with them and simply ignore them, which can make it difficult the next time an oral fluid collection is needed.

Antibiotic Screening

Oral fluid also holds promise as an antibiotic screening procedure, according to Locke Karriker, DVM, at Iowa State’s Veterinary Diagnostic and Production Animal Medicine department, and second year veterinary student Allison Meiszberg.

Currently, the only way to check an animal’s status for the presence of antibiotics is to test urine, serum or tissue, Karriker says.

That’s not a practical approach. But oral fluid could provide a pen-side method to screen animals for antibiotics prior to shipping them to slaughter, he says.

Karriker says the issue raises two questions in his mind: • Do antibiotics appear in oral fluid? • Do we have a handy test that would detect those antibiotics if they were present in oral fluid?

In a very preliminary pilot study, Karriker and Meiszberg used a very sensitive, rapid antibiotic test that screens bulk tank milk for antibiotics (snap test) to test oral fluid collected from four pens of 100-lb. pigs, each containing three pigs.

None of the pigs had received antibiotics. Two pens of pigs were injected with tetracycline, while the other two groups received ceftiofur sodium at labeled doses, Meiszberg explains. Then oral fluid samples were collected at 2, 6, 12 and 24 hours after injection.

The group of pigs given ceftiofur sodium was uniformly negative on oral fluid prior to receiving the antibiotic injection, and uniformly positive after being given the antibiotic when tested at all time points after injection, Karriker says. The results were similar but not as clear-cut for the tetracycline treatment groups.

“The preliminary results are very, very promising that oral fluid might be a useful and convenient vehicle for detecting antibiotics in a population of pigs,” he observes.

Besides providing a safeguard against marketing a group of hogs with potentially violative levels of antibiotic residues, the process also could be validated to assure notification if the antibiotics are being delivered to the wrong bins or animals are being medicated at incorrect levels, Karriker says.

For the first time in this decades-long struggle, pork producers will be in charge of their own destiny, he says.

“Every successful disease elimination program has accomplished one thing: don’t move the pigs until you know their status, and with oral fluid collection, that is the lynchpin where now we can do that,” he says.

Instead of relying on costly, labor-and-animal-intensive blood testing, producers are empowered to do their own survey sampling and collection in their barns before buying or selling pigs, Henry remarks.

“We can keep all those extra folks out of the barns doing blood testing, and for $50 on each end, those folks buying and selling pigs are physically able to get populations of animals identified and characterized for PRRS,” he adds.

Henry is chairman of the stakeholder group that oversees the PRRS Coordinated Agricultural Project that has funded Iowa State University’s PRRS research efforts, in coordination with the National Pork Board.

Conclusions

Zimmerman cautions this endorsement of oral fluid collection doesn’t diminish the value of blood testing as a confirmatory test or for use in individual testing situations. Oral fluid collection is another tool in the herd health management toolbox.

But he adds that this relatively inexpensive surveillance method offers a ready means of identifying disease interactions on hog farms, and is a new tool to finally begin to understand the whole swine disease picture in many herds, Zimmerman asserts.

“We need to start targeting our interventions earlier and smarter so that we can avoid disease problems and improve the bottom line,” he concludes.